Genet. Res., Camb. (2017), vol. 99, e2.

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doi:10.1017/S001667231600015X

Effective experimental validation of miRNA targets using an

improved linker reporter assay

CHEOLWON CHOI 1 † , JAMES HAN 2 † , NGUYEN THI THAO TRAN 1 , SEULGI YOON 1 ,
GOEUN KIM 1 , SUJUNG SONG 1 , YOUNGJO KIM 1 A N D SEONGHO RYU 1 *
1
Soonchunhyang Institute of Med-bioscience (SIMS), Sunchunhyang University, Chonan-Si, 336-745, Republic of Korea
2
Great Oak High School, Temecula, CA, USA
(Received 30 May 2016; revised 26 September 2016; accepted 7 November 2016)

Summary
miRNAs are small, non-coding RNAs that play critical roles in various cellular processes. Although there are
several algorithms that can predict the potential candidate genes that are regulated by a miRNA, these algo-
rithms require further experimental validation in order to demonstrate genuine targets of miRNAs. Moreover,
most algorithms predict hundreds to thousands of putative target genes for each miRNA, and it is difficult to
validate all candidates using the whole 3′-untranslated region (UTR) reporter assay. We report a fast, simple
and efficient experimental approach to screening miRNA candidate targets using a 3′-UTR linker assay.
Critically, the linker has only a short miRNA regulatory element sequence of approximately 22 base pairs in
length and can provide a benefit for screening strong miRNA candidates for further validation using the
whole 3′-UTR sequence. Our technique will provide a simplified platform for the high-throughput screening
of miRNA target gene validation.

1. Introduction human mRNAs (John et al., 2004; Lewis et al., 2005),

miRNAs are single-stranded, small, non-coding
RNAs composed of approximately 18–25 nucleotides
(Zhong et al., 2012). miRNAs are derived from pri-
miRNA, which has a hairpin-like structure and under-
goes subsequent cleaving processes to make mature
miRNA by Drosha and Dicer (Lee et al., 2006).
miRNAs have inhibitory functions for modulating the
post-transcriptional regulation of gene expression by par-
tial or complete binding to the 3′-untranslated region
(UTR) of their target mRNAs, thereby mediating
mRNA translational inhibition or degradation (Ha &
Kim, 2014). On the basis of bioinformatics analyses, indi-
vidual miRNAs are able to bind up to several hundred
genes, and miRNAs potentially regulate a third of
* Corresponding author: Dr. Seongho Ryu, Soonchunhyang
Institute of Med-bio Sciences (SIMS), Chonan-Si, 336-745,
Republic of Korea. Tel: +82 41 530 4839. E-mail: ryu@sch.ac.kr
†
These authors contributed equally to this work.
implying that miRNAs may impact all aspects of cell
biology. Indeed, miRNAs play important roles in
various biological processes, such as development, prolif-
eration, differentiation, cell fate determination, apop-
tosis, signal transduction, host–viral interaction and
tumorigenesis (Stern-Ginossar et al., 2007; Welch et al.,
2007; Choy et al., 2008; Ivey & Srivastava, 2010, Baum-
johann & Ansel, 2013). Dysregulation of miRNA–target
gene interaction can result in debilitating diseases,
including cancer development and metastasis (Tavazoie
et al., 2008; Hayes et al., 2014).
Despite multiple computational tools being useful
in predicting miRNA target genes, candidate genes
should be experimentally validated in order to obtain
full legitimacy. Conventional molecular techniques
such as northern blot, reverse transcription polymer-
ase chain reaction, microarrays, sequencing of RNA,
western blot and enzyme-linked immunosorbent
assay have been used to validate target genes. Never-
theless, these methods are not appropriate for validat-
ing the entire lists of candidate genes predicted from
C. Choi et al. 2

Fig. 1. Scheme of a modified linker reporter assay. According to the target prediction algorithms, including TargetScan,
miRanda and PicTar, the full sequences of 3′-untranslated regions (UTR) were analysed in order to find miRNA
regulatory element (MRE) sequences that putatively bind to miR-708. For the experimental validation, either the whole
3′-UTR (left), the MRE with a partial sequence of the 3′-UTR (middle) or only MRE sequences (right) were incorporated
into the reporter vector. Two cases – whole 3′-UTR (left) and only MRE sequences (right) – were tested in this study.

computational algorithms due to the limitations of
these techniques in terms of revealing direct correla-
tions between an miRNA and a candidate gene.
A 3′-UTR reporter assay has been commonly used
in order to experimentally validate direct miRNA tar-
gets (Aldred et al., 2011). This method is based on the
idea that miRNAs function by mediating translational
repression of their target mRNAs. Many studies have
been performed using in vitro 3′-UTR reporter assays
in order to validate the target genes of specific
miRNAs (Kuhn et al., 2008; Thomson et al., 2011).
Furthermore, a luciferase reporter system has been
recently utilized in order to study whether miRNAs
bind to the 3′-UTR regions of target genes (Oh &
Do Won Hwang, 2013).
To apply the luciferase reporter system, the 3′-UTR
regions of potential miRNA target genes have to be
cloned into a reporter vector. Traditionally, the
whole 3′-UTR region of a target gene was cloned for
use in the luciferase reporter system (Fig. 1). Even
though the whole 3′-UTR sequence is ideal for study-
ing an miRNA and its targets, it can take time and
effort to clone it. Recently, the use of only a surround-
ing region of the miRNA regulatory element (MRE)
(∼60 bp) was successful at validating the target genes
(Jin et al., 2013). This study used a chemically
synthesized ‘linker’ instead of amplifying the whole
3′-UTR region in order to validate candidate genes.
The ‘linker’ has a duplex form and contains the pre-
dicted MRE from computational algorithms and
restriction sites at both ends. Even though this study
proved that the surrounding region of the MRE is
sufficient for validating candidate genes, the linker
took a relatively long time to synthesize and the clon-
ing efficiency was low because the linker had the same
restriction enzyme at both ends.
Here, we describe the improved luciferase 3′-UTR
reporter assay containing only a short MRE sequence
(∼22 bp) and demonstrate that our new, improved
technique provides a simplified platform for validating
target genes compared with the conventional lucifer-
ase reporter assay.
2. Materials and methods
2.1. Primer and linker design
Lists of putative target genes are obtained from
miRNA target prediction tools such as miRanda,
TargetRank and TargetScan (Ryu et al., 2013).
MRE sequences are gained from the 3′-UTR
sequences of putative target genes by analysing
High-throughput validation of miRNA targets 3

Fig. 2. Designing a linker. (a) Either a miRNA regulatory element linker or whole 3′-untranslated regions of target genes
regulated by miR-708 were incorporated into the pmirGLO Dual Luciferase vector. Two different enzymes – NheI and
XbaI – are used for cloning. (b) The ‘linker’ is derived from self-ligation of two synthetically designed, single-stranded
primers. When two primers form a duplex, both ends become sticky ends automatically (5′-NheI and 3′-XbaI) and bind
directly to digested vectors without further enzyme digestion.

predicted miR-708 binding sequences. Linker
sequences are designed to have computationally pre-
dicted MRE sequences that can complementally
bind to miRNA (Fig. 2(b)). Two short, single-
stranded primers were chemically synthesized and
solubilized in 10 mM Tris (Qiagen, Venlo, The
Netherlands) as 100 μM stock (Table 1). Two paired
primers were self-ligated to form a double-stranded
linker. Simply put, each primer was mixed in a ratio
of 1:1 and boiled at 95 °C for 5 minutes and slowly
cooled to room temperature.
To ligate with a reporter vector, a linker should
have restriction enzyme sites at both ends. In our
method, the linker has two different restriction
enzyme sites: NheI and XbaI at the 5′ and 3′ ends of
the MRE sequence, respectively (Fig. 2(b)). Simply
put, when two primers form a duplex, both ends are
sticky and automatically mimic enzyme-digested
inserts. There is no extra restriction enzyme digestion
step. Sequence information is shown in Table 1 and
Table 2. All linkers were mixed in a single tube and
ligated with a vector at the same time. Vectors and lin-
kers were mixed in a ratio of 1:3 for ligation. T4 DNA
ligase enzyme was used for the ligation. Three-fold
greater numbers of colonies than numbers of linkers
were selected in order to determine the type of linker.
2.2. Construction of the linker reporter vector
pmirGLO Dual Luciferase vector was purchased
(Promega, Fitchburg, WI) and used to make the
modified luciferase reporter assay system. This vector
contains firefly luciferase (Fluc), which can be used to
measure the interactivity of miR-708 as described pre-
viously (Ko et al., 2009). Renilla luciferase (Rluc) is
used to normalize the efficiency of the transfection
of the DNA vector (Ko et al., 2009). Either the ‘linker’
or the whole 3′-UTR region of the target genes that
are regulated by miR-708 were incorporated into the
pmirGLO vector (Fig. 2(a)).
2.3. DNA transfection and luciferase activity assay
Transfection of the reporter vector was carried out using
a mixture of Lipofectamine 2000 (Invitrogen, Carlsbad,
CA) with reduced serum medium, Opti-MEM (Thermo
Scientific, Waltham, MA) in 293 T cells according to the
manufacturers’ instructions. Either ‘linker reporter
plasmid vector’ or empty vectors with or without miR-
708 were transfected into 293 T cells. A total of 1 μg of
each MRE linker reporter plasmid and empty or miR-
708-bearing vector was used for transfection into 293
T cells with 4 μl of Lipofectamine 2000. After 24-hour
incubations, 293 T cells were lysed with 1X passive
lysis buffer and luciferase activity was measured follow-
ing manufacturer’s instructions (Fig. 1).
3. Results
We previously reported the characteristic functions
of miR-708 and revealed its biological role in
cancer metastasis by regulating NNAT expression
C. Choi et al.
Table 1. Linker sequence information of putative miR-708 target genes.
Target gene Linker – forward
CXCL12 5′-TCGAGCCTCTACTAACAATCAGCTCCTTT-3′
CD44 5′-TCGAGAGAAAGAAGAAGAAAAGCTCCTGT-3′
ERBB4 5′-TCGAGGTTAGACTACCAAGCAGCTCCTGT-3′
ENAH 5′-TCGAGAATCAACATACTATAAGCTCCTGT-3′
GSK3A 5′-TCGAGGGCCATAGCCCATCAAGCTCCTGT-3′
FLT1 5′-TCGAGATATTAAGCACTTTAAGCTCCTTT-3′
NNAT 5′-TCGAGCCCAGCTAGATTGTAAGCTCCTTT-3′
FRS2 5′-TCGAGCCTCTTACACCAGTCAGCTCCTTT-3′’
EFNA5 5′-TCGAGCACCAGTGGTTCGTCAGCTCCTGT-3′
UNK-2 5′-TCGAGCACCTGGGCCCTTGAAGCTCCTTT-3′
CHERP 5′-TCGAGCAGAGCCGGCACCCCAGCTCCTTT-3′
USP9X-1 5′-TCGAGCATGGCAGTTGGATCAGCTCCTTT-3′
THRB-2 5′-TCGAGCTGGTTAGATCCTCAAGCTCCTTT-3′
MLL 5′-TCGAGTCCTTCCCCTATTGAAGCTCCTCT-3′
CKAP2 5′-TCGAGGCATTCACGGCAGTGAGCTCCTTT-3′
TLK1 5′-TCGAGGATATTGACTAACTGAGCTCCTTT-3′
NFAT5 5′-TCGAGTTTTGCCTCTATTCCAGCTCCTTT-3′
Table 2. Primer sequence information for cloning whole 3′-untranslated regions of putative miR-708 target genes.
Target gene Untranslated region – forward
CXCL12 5′-GTTTTCAGTGGGGCAGTCAT-3′
CD44 5′-GAACTTCCAAAGGCTGCTTG-3′
ERBB4 5′-TTCCAGAGGCCAATTGTAGC-3′
ENAH 5′-AACATTTGCACCCCATGAAT-3′
GSK3A 5′-CACCCTTCCACTTCCATCTG-3′
FLT1 5′-TCCCAGCTCTGACCCTTCTA-3′
NNAT 5′-GCCCCAGCTCCCAGCCCTGG-3′
(Ryu et al., 2013). NNAT was identified as a true tar-
get gene using a reporter vector with a whole 3′-UTR
region of NNAT, and this was confirmed by using a
reporter vector with a mutated binding site (Ryu
et al., 2013). In this study, we have utilized NNAT
as a confirmed target gene of miR-708 in order to
test our newly developed technique.
We obtained a list of predicted target genes, includ-
ing NNAT, by using several miRNA target prediction
algorithms such as miRanda, TargetRank and
TargetScan (Ryu et al., 2013). NNAT and HOXB3
are predicted by all three distinct algorithms (Ryu
et al., 2013). In addition, SSRP1, HNRNPK,
EPDR1 and FLT1 have been predicted by more
than two algorithms in our previous research (Ryu
et al., 2013).
Based on the results, we designed the appropriate
primers to amplify the whole 3′-UTR and to synthe-
size the ‘linker’ for a given list of candidate target
genes (Fig. 1). The lengths of the 3′-UTR regions var-
ied from several hundred to nearly 1000 bp (Table 2).
We then cloned them into the luciferase reporter vec-
tor, the pmirGLO Dual Luciferase vector. This vector
contains two independent luciferases: firefly luciferase
(Fluc) for the translational suppression and Renilla
luciferase (Rluc) for the normalization (Fig. 2).
4
Linker – reverse
5′-CTAGAAAGGAGCTGATTGTTAGTAGAGGC-3′
5′-CTAGACAGGAGCTTTTCTTCTTCTTTCTC-3′
5′-CTAGACAGGAGCTGCTTGGTAGTCTAACC-3′
5′-CTAGACAGGAGCTTATAGTATGTTGATTC-3′
5′-CTAGACAGGAGCTTGATGGGCTATGGCCC-3′
5′-CTAGAAAGGAGCTTAAAGTGCTTAATATC-3′
5′-CTAGAAAGGAGCTTACAATCTAGCTGGGC-3′
5′-CTAGAAAGGAGCTGACTGGTGTAAGAGGC-3′
5′-CTAGACAGGAGCTGACGAACCACTGGTGC-3′
5′-CTAGAAAGGAGCTTCAAGGGCCCAGGTGC-3′
5′-CTAGAAAGGAGCTGGGGTGCCGGCTCTGC-3′
5′-CTAGAAAGGAGCTGATCCAACTGCCATGC-3′
5′-CTAGAAAGGAGCTTGAGGATCTAACCAGC-3′
5′-CTAGAGAGGAGCTTCAATAGGGGAAGGAC-3′
5′-CTAGAAAGGAGCTCACTGCCGTGAATGCC-3′
5′-CTAGAAAGGAGCTCAGTTAGTCAATATCC-3′
5′-CTAGAAAGGAGCTGGAATAGAGGCAAAAC-3′
Untranslated region – reverse Size (bp)
5′-ACACACAGCCAGTCAACGAG-3′ 539
5′-GGAGTGGCTTGTTGCTTTTC-3′ 550
5′-AGCAGAGCCTCACACAGTCC-3′ 573
5′-TGCATGCAATTCACACAGAG-3′ 560
5′-GGAGGTCTGTACACAGGCAAA-3′ 566
5′-CCCTTTCCTACCCTGTCTCC-3′ 824
5′-GTGCGCCTCTACTGCACCGC-3′ 925
To make the linkers, we chemically synthesized two
short sequences of ‘linker’ in duplex form, having
restriction enzyme sites at both ends of the MRE
sequence, as is shown in Table 1. In this study, we used
different restriction enzymes – NheI and XbaI – for
each end in order to prevent self-ligation of the vector
without a dephosphorylation step (Fig. 2(b)), which
was used in the previous method employing a surround-
ing MRE region (Jin et al., 2013).
First, we tested whether our newly developed tech-
nique was accurate enough to suppress luminescence
signals by miRNAs in comparison with the conven-
tional luciferase reporter assay. We subsequently mea-
sured luciferase activity after the transfection of either
empty or miR-708-expressing vectors in 293 T cells.
Importantly, the luciferase activity linked to the
NNAT linker was dramatically decreased after trans-
fection of miR-708-expressing vector compared to
empty vector (Fig. 3(a)). Critically, we demonstrated
the validated target gene NNAT as being directly
regulated by miR-708 using a linker reporter assay.
We then compared the luciferase activity between
the full lengths of the 3′-UTR regions and a MRE
linker for the various candidate target genes of miR-
708. However, the other genes were not heavily
affected by the transfection of miR-708, which is
High-throughput validation of miRNA targets 5

Fig. 3. Modified linker reporter assays are comparable to the whole 3′-untranslated region (UTR) reporter assay. Various
candidate genes, including NNAT and CD44, were predicted by several target prediction algorithms and selected for 3′-
UTR cloning. (a) Either empty or miR-708-bearings vector were co-transfected with a reporter vector into 293 T cells.
After 24 hours of incubation, the activity of luciferase was measured. The luciferase signals from the whole 3′-UTR were
compared with the signals from a miRNA regulatory element (MRE) linker. (b) Many linkers were selected for high-
throughput miRNA target assay. The MRE sequences were designed by the Probability of Interaction by Target
Accessibility (PITA) target prediction algorithm and cloned into the reporter vector. Either empty or miR-708-bearing
vectors were co-transfected with a reporter vector into 293 T cells. After 24 hours of incubation, luciferase activity was
measured. *p-value < 0·05.

consistent with the previous conventional luciferase
reporter assay (Fig. 3(a)). The results of both the con-
ventional 3′-UTR and the linker luciferase assay con-
sistently discriminated genuine targets (e.g., NNAT
and CD44) from a list of candidate target genes of
miR-708. Taken together, these data suggest that
our newly developed technique is reliable for validat-
ing target genes relative to conventional methods.
We found that NNAT and CD44 are the true tar-
gets of miR-708 by both conventional 3′-UTR and
linker luciferase assays, as previous studies have
shown (Saini et al., 2012; Ryu et al., 2013). To find
novel, genuine targets of miR-708, we further in-
vestigated the candidate target genes of miR-708
using another prediction algorithm, Probability of
Interaction by Target Accessibility (PITA). This algo-
rithm had been developed to predict the target genes
of miRNAs based on target gene accessibility and
similarity (Kertesz et al., 2007).
According to the PITA algorithm, a total of 2069
genes were predicted as target candidates. A total of
45 genes were predicted by both PITA and the in-
C. Choi et al.
house algorithm, which only considers the maximum
similarity value. We then selected 16 genes out of
45, including MLL and TLK1, for further validation
(Fig. 3(b)). Even though several genes, including
TLK1, EFNA5 and SSH-2, were suppressed (approxi-
mately 20% by miR-708), suppression was not as large
compared with the suppression of NNAT, which was
close to 50% (Fig. 3(b)). To confirm whether or not
these signals were noise, we assessed another
miRNA, miR-200a. As we expected, miR-200a does
not suppress the luciferase activity of NNAT, SSH-2,
EFAN5 and TLK1 linkers (Supplemental Figure 2(a)
& 2(b)). By combining these data, we suggest that
our new, modified linker reporter assay selectively
suppresses the targets of miRNA dependent on their
complementary binding.
4. Discussion
Bioinformatics tools facilitate the study of the charac-
teristic functions of miRNAs by providing a list of
potential target genes (Krek et al., 2005; Lewis
et al., 2005; Ryu et al., 2013). However, the currently
available computational methods and techniques
make it difficult to validate the entire list of hundreds
to thousands of target genes obtained from target pre-
diction algorithms due to their demands on time and
effort. Recently, luciferase reporter assays have been
widely used to monitor miRNA and validate target
genes linked to the 3′-UTR (Kuhn et al., 2008;
Thomson et al., 2011; Oh & Do Won Hwang,
2013). However, it is inefficient to clone each 3′-
UTR region of hundreds of target genes.
In this study, we describe an improved linker
reporter assay and have observed that this linker
reporter assay produces data that are consistent
with conventional reporter assays using whole 3′-
UTR regions (Fig. 3(a)). These results indicate that
our protocol is applicable for validating target genes.
In addition, our improved linker reporter assay has
economic advantages compared to previous reporter
assays containing surrounding MRE regions (Jin
et al., 2013). The linker is only 18–22 bp in length, cor-
responding to the predicted miRNA MRE sequence,
in contrast to the previous protocols containing linkers
of 60 bp in length for the surrounding MRE region
(Jin et al., 2013). It is cost effective since shorter
sequences can be chemically synthesized at lower costs.
We further tested several candidate genes, including
NNAT, CD44, SSRP-1, TLK1, EFNA5, by using west-
ern blot analysis, but only NNAT showed suppressed
signals (Supplemental Figure 2(c)). These data suggest
that a further step using western blot analysis is neces-
sary in order to validate the real target of an miRNA. It
is possible that some MRE sequences are not accessible
to miRNAs because a whole 3′-UTR sequence forms a
6
complicated secondary structure, and so some MREs
are not accessible to miRNAs. Nevertheless, our
method can narrow the entire list of candidate targets
from several hundreds to few strong candidates.
These strong candidates will be further validated with
western blot analysis at the protein level.
Previous protocols also performed an extra depho-
sphorylation step after cutting the vector in order to
prevent self-ligations (Jin et al., 2013). However, our
protocol does not require this step because the linker
contains two different restriction enzyme sites at
each end. Furthermore, our new, modified reporter
assay also has outstanding advantages for designing
mutated miRNA binding sites for the further valid-
ation of target genes. Taken together, our technique
is a cost-effective and efficient method compared to
the previous protocols that have been performed in a
high-throughput manner.
Our technique will be applied not only for the valid-
ation of the target genes of miRNAs, but also for mon-
itoring miRNA biogenesis in various experimental
conditions in vivo. Thus, the study of miRNA biogenesis
may allow us to understand more deeply the biological
processes and cellular mechanisms of organogenesis
and cell differentiation. Collectively, our approach is
efficient for validating candidate miRNA target genes
and may provide an opportunity to discover novel
genes and cellular mechanisms as potential prognosis/
diagnosis markers and therapeutic targets.
This research was supported by the Marine Biotechnology
Program (PJT200620) funded by the Ministry of Oceans
and Fisheries and the Basic Science Research Program
through the National Research Foundation of Korea
(NRF) funded by the Ministry of Science, ICT & Future
Planning (2014R1A1A1006650), Korea.
5. Declaration of interest
The authors have no commercial or financial conflicts
of interest.
6. Authors’ contributions
J. Han, C. Choi and S. Ryu conceived and designed the
experiments. J. Han, C. Choi, T. Nguyen, S Yoon and
S. Ryu performed the experiments. S. Song and
Y. Kim analysed the data. J. Han, C. Choi, Y. Kim
and S. Ryu wrote the manuscript. All authors contrib-
uted to critically revising the manuscript for important
intellectual content, gave final approval and agreed to
be accountable for all aspects of the work.
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